[Electroacupuncture regulates blood glucose dysregulation by up-regulating IGF1-mediated PI3K/Akt signaling pathway in mice with circadian rhythm disorder].

Objectives: To investigate the mechanism of electroacupuncture (EA) underlying improvement of abnormal blood glucose in mice with disturbance of peripheral biological clock.

Methods: C57BL/6J mice (half male and half female) were randomly divided into normal control (n=26), model (n=26), EA (n=26) and EA+ inhibitor (n=8) groups. The circadian rhythm disturbance model was established by subjecting the mice to constant light (12-h light-light [LL] cycle) for 4 weeks. EA (4 Hz/20 Hz, 0.2 mA) was applied to bilateral "Ganshu" (BL18) for 15 min, once daily for 8 weeks. The mice of the EA+inhibitor group were given LY294002 (40 μmol/L, 10 μL) by intraperitoneal injection for blocking activities of phosphatidylinositol-3-kinase (PI3K) signaling pathway, once every other day for 8 weeks. After the intervention, the fasting plasma glucose (FPG) was measured, and fasting serum insulin (FINS) and the circadian rhythm of liver insulin-like growth factor 1 (IGF1) were observed by measuring their contents with ELISA. The insulin resistance was evaluated by homeostasis model assessment of insulin resistance (HOMA-IR) and quantitative insulin sensitivity check index (QUICKI). The histopathological changes of the liver tissue were observed by H.E. staining. The changes of liver glycogen granule deposition were observed by PAS staining. The immunoactivity of forkhead box protein O1 (FoxO1) and glycogen synthase kinase 3β(GSK3β) in the liver tissue was detected by immunofluorescence technique. The expression levels of IGF1, IGF1R, FoxO1, GSK3β, phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6 phosphatase(G6Pase) mRNA and protein kinase B (Akt) and phosphorylated (p)-Akt proteins in the liver tissue were detected by qPCR and Western blot, respectively.

Results: In comparison with the normal control group, daily water intake, the contents of FPG, FINS and HOMA-IR, the immunoactivity of GSK3β and FoxO1 in liver tissue, and the expression levels of IGF1R, GSK3β, FoxO1, PEPCK and G6Pase mRNA in liver tissue were significantly increased (P<0.01, P<0.05), while the serum QUICKI, ratio of p-PI3K/PI3K and p-Akt/Akt, and expression of IGF1 mRNA in liver tissue obviously decreased (P<0.01) of mice in the model group. Compared with the model group, all the above indicators were significantly reversed in the EA group (P<0.05, P<0.01). After administration of the inhibitor LY294002 of PI3K signaling, the effects of EA in up-regulating the ratio of p-PI3K/PI3K and p-Akt/Akt, and in down-regulating the immunoactivity of GSK3β and FoxO1, and the expression levels of GSK3β, FoxO1 and G6Pase mRNA in liver tissue were eliminated (P<0.05). H.E. staining showed irregular arrangement of the hepatocytes with diffuse swelling, loose connections between hepatocytes, fat vacuoles of different sizes in the cytoplasm, and diffuse steatosis in some mice of the model group. PAS staining showed disordered arrangement of hepatocytes, with a large number of fat vacuoles, and relatively thin and uneven staining between and within cells in the model group. These situations were evidently improved in the EA group and EA+ inhibitor group, including reduction in the arrangement of liver cells and the vacuoles of fat in cytoplasm.

Conclusions: Acupuncture of BL18 can not only improve the disordered circadian rhythm, but also lower the abnormally elevated blood glucose in mice with disturbance of circadian rhythm, which may be related to its functions in activating IGF1-mediated PI3K/Akt signaling, thereby inhibiting the mRNA and protein expression of GSK3β, FoxO1, PEPCK and G6Pase, and improving liver glucose metabolism.